The mammalian immune system provides a means for the recognition and elimination of foreign pathogens. While the immune system normally provides a line of defense against foreign pathogens, there are many instances where the immune response itself is involved in the progression of disease. Exemplary of diseases caused or worsened by the host's own immune response are autoimmune diseases such as multiple sclerosis, lupus erythematosus, psoriasis, pulmonary fibrosis, and rheumatoid arthritis and diseases in which the immune response contributes to pathogenesis such as atherosclerosis, inflammatory diseases, osteomyelitis, ulcerative colitis, Crohn's disease, and graft versus host disease often resulting in organ transplant rejection.
Macrophages are generally the first cells to encounter foreign pathogens, and accordingly, they play an important role in the immune response. However, activated macrophages can contribute to the pathophysiology of disease in some instances. Activated macrophages nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response.
Rheumatoid arthritis (RA) is a systemic disease characterized by chronic inflammatory synovitis, usually involving peripheral joints. The synovial inflammation causes cartilage deterioration and bone erosion with consequent destruction of joint integrity. Rheumatoid factors, which are autoantibodies reactive with the Fc region of IgG, are found in more than two-thirds of patients with RA indicating that RA has an autoimmune component.
RA is seen throughout the world in as much as 2% of the population, with 80% of RA patients developing the disease between the ages of 35 and 50. The clinical manifestations of RA include pain, swelling, and tenderness in the joints resulting in limitation of motion, weakness, fatigue, and weight loss. RA is a systemic disease and, consequently, has extra-articular manifestations, especially in patients with high titers of rheumatoid factors. These symptoms include rheumatoid nodules with an inner zone of necrotic material, a mid-zone of macrophages, and an outer zone of granulated tissue, muscle atrophy, osteoporosis, pulmonary fibrosis, and rheumatoid vasculitis which may result in cutaneous ulceration, digital gangrene, or neurovascular disease.
Rheumatoid synovitis, characteristic of RA, results in an increase in the number of synovial lining cells, hyperplasia and hypertrophy of the synovial lining cells, microvascular injury, edema, and infiltration of cells such as T cells, macrophages, and dendritic cells. The rheumatoid synovium is characterized by the presence of secreted products of immune cells such as factors secreted by T lymphocytes including IL-2, IFN-δ, IL-6, IL-10, GM-CSF and TGFα and β and factors secreted by activated macrophages including IL-1, IL-6, IL-8, IL-10, GM-CSF, macrophage CSF, and TGFβ. The production of these cytokines appears to account for much of the pathology of RA including inflammation of the synovium, synovial cell proliferation, cartilage and bone deterioration, and systemic symptoms of the disease.
RA may be treated using various therapies including physical therapy, rest, and splinting. Therapeutic agents are also used for the treatment of RA including aspirin and nonsteroidal anti-inflammatory drugs to control local inflammation. However, these agents have a minimal effect on the progression of the disease and are associated with toxic side effects. Disease-modifying anti-rheumatic drugs, such as α-penicillamine and sulfasalazine, are also used to treat RA, but the benefit from these drugs is delayed for weeks or months and these drugs have toxic side effects. Immunosuppressive and cytotoxic drugs suppress symptoms of RA in some patients, but are associated with toxicity. Intra-articular glucocorticoids have also been used, but provide only transient relief. Accordingly, there is a need for the development of new therapies with reduced toxicity that are efficacious for the treatment of RA and other diseases caused or worsened by activated macrophages.
The folate receptor (FR) is a 38 KDa GPI-anchored protein that binds the vitamin folic acid with high affinity (<1 nM). Following receptor binding, rapid endocytosis delivers the vitamin into the cell, where it is unloaded in an endosomal compartment at low pH. Importantly, covalent conjugation of small molecules, proteins, and even liposomes to folic acid does not alter the vitamin's ability to bind the folate receptor, and therefore, folate-drug conjugates can readily enter cells by receptor-mediated endocytosis.
Because most cells use an unrelated reduced folate carrier (RFC) to acquire the necessary folic acid, expression of the folate receptor is restricted to a few cell types. With the exception of kidney and placenta, normal tissues express low or nondetectable levels of FR. However, many malignant tissues, including ovarian, breast, bronchial, and brain cancers express significantly elevated levels of the receptor. In fact, it is estimated that 95% of all ovarian carcinomas overexpress the folate receptor. It has recently been reported that FRβ, the nonepithelial isoform of the folate receptor, is expressed on activated (but not resting) synovial macrophages. Thus, Applicants have attempted to utilize folate-linked compounds potentially capable of altering the function of activated macrophages, to treat macrophage-mediated disease states. For example, Applicants have found that folate-linked immunogens can be used to redirect the host immune response in arthritic animals to activated macrophages at the site of inflammation to deplete macrophages and reduce arthritic inflammation.
Scintigraphic imaging agents are a million times more sensitive than magnetic resonance imaging (MRI) contrast agents, and their selectivity can be enhanced by their targeting to lesion-specific cell markers. Indeed, the radioisotope 99mTc has been delivered to arthritic tissues using nonspecific IgG, anti-CD4 antibodies, CD11b/CD14-glycolipopeptide ligands, and E-selectin binding peptides. Preclinical studies with such radioimaging agents have clearly emphasized the value of imaging arthritic tissues in-vivo, however, the selectively of the current imaging agents is not yet optimal, and none of the present compounds is targeted exclusively to activated macrophages. In view of the emergence of folate receptor activity during macrophage activation, Applicants have undertaken to determine whether a folate-targeted 99mTc imaging agent might be used to image arthritic lesions in vivo.
To determine whether expression of this high affinity FR might be exploited to selectively target drugs to activated macrophages at sites of inflammation, folic acid has been conjugated to a 99mTc chelator, and its distribution evaluated in both normal and diseased tissues of rats with adjuvant-induced arthritis. The folate-linked 99mTc chelate complex, termed EC20, was indeed found to concentrate in the arthritic extremities of diseased rats, but not in the joints of healthy rats. The intensity of the gamma scintigraphic images of affected tissues was found to be greatly reduced in the presence of excess competing folic acid. Furthermore, liver and spleen of arthritic animals also showed enhanced uptake of EC20 and increased levels of FR, confirming that systemic activation of macrophages accompanies adjuvant-induced arthritis. Depletion of macrophages from arthritic animals reduced tissue FR content and concomitantly abolished uptake of EC20. Furthermore, Kupffer cells isolated from rats with adjuvant-induced arthritis exhibited a significantly higher binding capacity for folate conjugates than Kupffer cells from healthy rats. Thus, Applicants have found that EC20 is useful for assaying the participation of activated macrophages in inflammatory pathologies such as rheumatoid arthritis.
The present invention is directed to a method for treating and monitoring disease states mediated by activated macrophages. In accordance with one embodiment of the present invention, disease states mediated by activated macrophages are treated by redirecting host immune responses to activated macrophages or by altering the function of activated macrophages or by direct killing of activated macrophages. In one aspect of the invention, to promote killing of activated macrophages, ligands that bind specifically to activated macrophages are conjugated with an immunogen to redirect host immune responses to the activated macrophage population, or they are conjugated to a cytotoxin for direct killing of macrophages. Ligands that can be used in the conjugates of the present invention include those that bind to receptors expressed specifically on activated macrophages, such as the folate receptor, or ligands such as monoclonal antibodies directed to cell surface markers specifically expressed on activated macrophages. In another aspect of the invention ligands that bind specifically to activated macrophages are conjugated with an imaging agent; the conjugate is administered to a patient for diagnosing and monitoring the progression of diseases mediated by activated macrophages.
In one embodiment, a method of treating or monitoring/diagnosing a disease state mediated by activated macrophages is provided. The method comprises the step of administering to a patient suffering from a macrophage mediated disease state an effective amount of a composition comprising a conjugate or complex of the general formula Ab-X, where the group Ab comprises a ligand capable of binding to activated macrophages, and when the conjugate is being used for treatment of the disease state, the group X comprises an immunogen, a cytotoxin, or a compound capable of altering macrophage function, and when the conjugate is being used for monitoring/diagnosing the disease state, X comprises an imaging agent.